Device and Method for Isolating Extracellular Vesicles From Biofluids

ABSTRACT

A device and method for isolating extracellular vesicles from biofluids is disclosed. A nanoporous silicon nitride membrane is provided with a tangential flow of biofluid. A pressure gradient through the nanoporous silicon nitride membrane facilitates capture of extracellular vesicles from the tangential flow vector of biofluid. Reversal of the pressure gradient results in the release of the extracellular vesicles for subsequent collection.

CROSS REFERENCE TO RELATED PATENT APPLICATIONS

This application claims priority to U.S. Patent Application Ser. No.62/443,749 filed Jan. 8, 2017 entitled “Device and Method for IsolatingExtracellular Vesicles From Biofluids” by Dr. James L. McGrath et al.,and to International Application Number PCT/US18/12735 filed Jan. 8,2018 entitled “Device and Method for Isolating Extracellular VesiclesFrom Biofluids”, the entire disclosures of which are incorporated hereinby reference in their entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under 1521373 awarded byNational Science Foundation. The government has certain rights in theinvention.

BACKGROUND OF TIE INVENTION 1. Field of the Invention

The present invention relates generally to fluid separation, and moreparticularly to a device and related method for separating extracellularvesicles from biofluids.

2. Description of Related Art

Extracellular vesicles, once thought to be simply membrane debris withno biological value, are now understood to play a vital role in cell tocell communication within multi-cellular organisms.

Extracellular vesicles are lipid bilayer particles derived from severalcellular pathways including exosomes, microvesicles, and apoptoticbodies. Exosomes of 30-100 nm diameter are derived from the endosomalpathway. Microvesicles of 100 nm-1 um diameter are derived from theplasma membrane. Extracellular vesicles can be found in biofluids suchas blood, plasma, serum, urine, cerebrospinal fluid, aqueous humor,lymph, breast milk, semen, and conditioned cell culture media, amongothers.

It is now known that extracellular vesicles have significance in normalphysiological processes including tissue regrowth and repair,immunological responses, coagulation of blood, and also in thepathological progress of many diseases. As such, they representtremendous possibilities for therapeutic applications. The applicationsfor extracellular vesicles are growing in diversity and significance forboth normal and pathological processes. Identifying specific EV markersoffers tremendous potential for new therapeutic targets, as well as forliquid biopsy prognostics and for companion diagnostics to monitortreatment response. Extracellular vesicles also can transport nucleicacids during cell to cell communication, and thus represent tremendouspotential for drug delivery vehicles. Extracellular vesicles also holdequally tremendous potential as therapeutics in regenerative medicine,as vaccination agents and as delivery agents by way of using theirinnate ability to transmit various RNA species among cells.

While the uses for extracellular vesicles continues to increase both inresearch and in direct and indirect therapeutic applications, isolatingextracellular vesicles from bodily fluids remains a difficult and slowprocess. Techniques such as ultrafiltration and gelation result insignificant contamination from protein and complex and tedious secondaryprocesses to eliminate the protein contamination from the desiredextracellular vesicles.

What is therefore needed is a device that captures the majority ofextracellular vesicles in a bodily fluid while avoiding proteincontamination. What is also needed is a device that is reusable andrelatively fast in capturing and retaining extracellular vesicles in abodily fluid.

BRIEF SUMMARY OF THE INVENTION

In accordance with the present invention, there is provided a device andmethod for isolating extracellular vesicles from biofluids. A nanoporoussilicon nitride membrane is provided and receives a tangential flow ofbiofluid. A pressure gradient through the nanoporous silicon nitridemembrane facilitates capture of extracellular vesicles from thetangential flow vector of biofluid. Reversal of the pressure gradientresults in the release of the extracellular vesicles for subsequentcollection. Defined surface chemistries of the nanoporous siliconnitride membrane may also be employed to augment capture and subsequentcollection of these extracellular vesicles.

The foregoing has been provided by way of introduction, and is notintended to limit the scope of the invention as described by thisspecification, claims and the attached drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will be described by reference to the following drawings,in which like numerals refer to like elements, and in which:

FIG. 1 depicts capture of exosomes on a tangential flow device of thepresent invention;

FIG. 2 depicts clearing of protein contaminants on the tangential flowdevice of FIG. 1;

FIG. 3 depicts collection of exosomes using transmembrane pressurereversal of the device of FIG. 1;

FIG. 4 is a schematic diagram of a microfluidic chip based device of thepresent invention;

FIG. 5 shows exosome capture from pure plasma on nanoporous siliconnitride;

FIG. 6 shows isolation of a sample prepared with a reagent andassociated membrane clogging;

FIG. 7 shows dead end centrifugation of plasma with NPN membrane andresulting protein fouling;

FIG. 8 depicts purification of exosomes using an NPN membrane intangential flow mode:

FIG. 9 depicts a supported membrane and associated exosome capture;

FIG. 10 depicts EDX chemical analysis of an in use membrane of thepresent invention; and

FIG. 11 is a diagram of fluid dynamics associated with exosome capture.

The present invention will be described in connection with a preferredembodiment, however, it will be understood that there is no intent tolimit the invention to the embodiment described. On the contrary, theintent is to cover all alternatives, modifications, and equivalents asmay be included within the spirit and scope of the invention as definedby this specification, claims and drawings attached hereto.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention involves the capture and physical retention andsieving of extracellular vesicles from biofluids. A device and methodfor isolating extracellular vesicles from biofluids is thus described,with various embodiments also described and envisioned herein.

The present invention makes use of nanoporous silicon nitride membranesin a tangential flow device, wherein the extracellular vesicles arecaptured by a novel, diffusion-driven, physical sieving mechanism,allowing for subsequent isolation and purification thereof.

In use, a biofluid is slowly passed over the nanoporous silicon nitridemembrane under conditions of slight negative transmembrane pressure.This configuration permits the diffusion of extracellular vesiclestoward the nanoporous membrane, such that the extracellular vesicles arecaptured in the pores of the membrane. While maintaining a negativetransmembrane pressure, the extracellular vesicles can be retained inthe pores while the fluid component of the biofluid is swept and clearedaway, thus removing unwanted constituents from the biofluid. Whilemaintaining transmembrane pressure, the captured extracellular vesiclescan be washed in a clean solution to increase their purity. Finally, thetransmembrane pressure can be released or reversed to slightly positiveand the isolated extracellular vesicles are eluted off the membrane in abolus of clean solution.

Surprisingly, extracellular vesicles are captured under nativeconditions using the present invention. Other methods require additionof buffers and/or salts to manipulate the pH or ionic strength of thebiofluid in order to absorb the extracellular vesicles onto a filtrationmedia for their isolation.

The physical sieving mechanism described herein where the extracellularvesicles are captured on the pores of the nanoporous silicon nitridemembrane by diffusion into the slight transmembrane pressure environmentof the porous membrane, in the context of a tangential flowconfiguration of the present invention, seems to depend on an excess ofpores relative to the number of extracellular vesicles in the biofluid.Thus, a large pore-to-extracellular vesicle ratio is required for theisolation mechanism of the present invention and will likely only workwith highly permeable membranes with a large density of pores (e.g., ˜;10⁷ pores per mm²).

The tangential flow configuration described herein results in theapparent removal of the unwanted but highly abundant species within mostbiofluids, with little residual contamination. For example, the highprotein content of plasma can be removed from captured extracellularvesicles so that a highly pure extracellular vesicle preparation isrealized.

For a more thorough understanding of the present invention and thevarious embodiments described and envisioned herein, reference is nowmade to the Figures.

FIG. 1 depicts capture of exosomes on a tangential flow device of thepresent invention. A tangential fluid flow device for creating atangential fluid flow velocity of a biofluid across the surface of thenanoporous membrane may include a conduit, a vessel, a channel, a tube,or any structure capable of retaining a fluid under flow conditions. Thetangential fluid flow in turn may be created using a pump, gravity,electrostatic forces, thermal or chemical gradients, or the like. Thevector labeled “plasma in” illustrates tangential flow across ananoporous silicon nitride (NPN) membrane where a pressure gradientexists, providing a slightly lower pressure below the membrane thanabove the membrane, which pulls extracellular vesicles such as exosomesinto the pores of the NPN membrane as protein is cleared. A pressuregradient from one side of the nanoporous membrane to the other side(through the nanoporous membrane) may be created with a pressuregradient device such as a pump, a diaphragm, a vacuum device, athermoelectric device such as a peltier device, a mechanical device forfluid flow modification, or the like. Further, a device for reversingthe created pressure gradient is provided, and may include a pumpcontroller for changing the direction of pump rotation, a switch forchanging polarity or removing electrical power from a thermoelectricdevice, a valve or fluid diversion structure, a vacuum controller forchanging or removing vacuum from one side of the membrane, or the like.As labeled in FIG. 1, the extracellular vesicles are diagrammaticallydepicted as shaded circles and protein is diagrammatically depicted as adistorted asterisk of sorts. The dotted line depicts the surface of theNPN membrane. Such a membrane is described, for example, in UnitedStates Patent application publication 2016/0199787 A1 to Striemer et al.and entitled Nanoporous Silicon Nitride Membranes, And Methods ForMaking And Using Such Membranes, the entire disclosure of which isincorporated herein by reference. Other membranes, devices and methodsapplicable to the present invention and the various embodimentsdescribed, depicted and envisioned herein are disclosed in U.S. Pat.Nos. 8,518,276 and 8,501,668, the entire disclosures of which areincorporated herein by reference in their entirety. Once theextracellular vesicles are captured through the process depicted anddescribed by way of FIG. 1, a cleanup stage occurs as depicted in FIG.2.

For exosome capture in the tangential flow device of the presentinvention, in a preferred embodiment, transmembrane pressure inoperation will be 1 pascal-1 atmosphere. Flow velocity will be 10μm/sec.-10 cm./sec. Channel length will be 1 mm.-1 m. along theprincipal direction of flow. A large channel size may be used, forexample in a large industrial size operation. Roll to roll processing,for example, could be used to create sheets of nanoporous siliconnitride (NPN). Channel height will be 100 nm.-1 mm. Pore diameter willbe 20 nm.-35 nm., or in some embodiments of the present invention, 20nm.-80 nm., or up to 120 nm. The membranes in some embodiments may be asthin as 30 nm.

FIG. 2 depicts clearing of protein contaminants on the device of thepresent invention. Once the extracellular vesicles are extracted fromthe plasma, a buffer solution is passed through the system to clearprotein contaminants, leaving behind extracellular vesicles entrapped orotherwise captured in the nanoporous silicon nitride (NPN) membrane. Thenext step, as described in FIG. 3, involves the collection of theentrapped extracellular vesicles using transmembrane pressure reversalsuch that the pressure gradient from one side of the nanoporous siliconnitride (NPN) membrane is reversed to then allow for the collection ofthe extracellular vesicles in a controlled volume. The area of thesilicon nitride membrane (NPN) can be varied to change the quantity ofextracellular vesicles collected in each pass or cycle of plasma flow,buffer rinse and pressure reversal. One can envision such a cycle to berepeated many times over in proportion to plasma flow and quantity.

Packaging of the device may include industrial or laboratory scalesetups, or may, in some embodiments of the present invention, includechip based packaging. For example, FIG. 4 is a schematic diagram of amicrofluidic chip based device of the present invention. Microfluidicaccess at the inlet and outlet of the top and bottom chambers can beseen.

FIG. 5 shows exosome capture from pure plasma on nanoporous siliconnitride where extracellular vesicles are trapped in NPN pores. The insetin FIG. 5 depicts captured extracellular vesicles adjacent to open poresin the NPN membrane.

There may be a variety of pore geometries employed. For example, a “bowlwith a hole structure where the side of the pore facing the tangentialflow of the material to be processed has a wide mouth and the back sidehas a smaller hole than the side facing the tangential flow. Thisgeometry allows the liquid to drain through the smaller hole whilepreventing escape of the exosome in the tangential flow. Such a geometrymaximizes the range of exosome capture while still allowingtransmembrane flow. In other words, while the nanoporous membranecomprises a first surface having a plurality of pores that are exposedto the tangential flow of material, the nanoporous membrane furthercomprises a second surface where the plurality of pores from the firstsurface extend through the second surface and where the plurality ofpores of the first surface are of a greater diameter than the relatedplurality of pores that extend through the second surface.

FIG. 6 shows isolation of a sample prepared with Exoquick™ reagent andassociated membrane clogging. The contamination of the NPN membrane bythe ExoQuick™ polymer is evident from the texture and electron beamcharging effects (shading). Note also that fewer exosomes are seen inthe image and many pores appear clogged. The ability of NPN to purifyextracellular vesicles such as exosomes directly from raw biofluids likeplasma promises both a simpler and cleaner preparation of exosomes.

Examples of devices according to the present invention that can performextracellular vesicle isolation as described herein include microfluidicformat devices.

By way of example, and not limitation, one can envision a two-channelconfiguration with the semi-permeable nanoporous membrane fluidicallyconnecting the two flow channels. In this embodiment, the nanoporousmembrane could be nanoporous silicon nitride (NPN) with ˜30 nm diameterpores. This membrane would permit the capture of exosomes that are30-100 nm diameter. Such material is described, for example, inPCT/US2014/1051316, the entire disclosure of which is incorporatedherein by reference.

In another embodiment, a second semi-permeable membrane, with 100, 200or 1,000 nm diameter pores is placed upstream of the first 30 nmdiameter pore membrane and fluidically connected so that extracellularvesicles smaller than the pores of the first membrane permeate to thesecond membrane with ˜30 nm pores. This would permit size fractionationof a biofluid's extracellular vesicles into micro vesicle (>100 nm but<1.000 nm) and exosome (30-100 nm) fractions.

FIG. 7 shows dead end centrifugation of plasma with a nanoporous siliconnitride membrane and resulting protein fouling. The resulting protein“cake” precludes separation of extracellular vesicles from the plasma,and dramatically illustrates why ultrafiltration and gelation methodsare unable to purify exosomes from plasma without significantcontamination from protein. The high permeability of ultrathinnanoporous silicon nitride membranes however, in a tangential flowconfiguration as described herein, remain clear of protein fouling andcake buildup. While not wishing to be bound to any particular theory,one hypothesis is that the high permeability of ultrathin nanoporoussilicon nitride allows for enough transmembrane flow to pull exosomesinto the pores of the nanoporous silicon nitride while in turn keepingthe nanoporous silicon nitride membrane free of protein cake buildup.

FIG. 8 depicts purification of exosomes using a nanoporous siliconnitride membrane in tangential flow mode, as has been previouslydescribed herein. Note that the pores of the membrane are visible afterpassing undiluted plasma over the membrane indicating the absence ofprotein fouling with the present invention despite exposure toremarkably high protein levels. The bright ‘glow’ of the sphericalparticles is characteristic of organic materials on the inorganicnanoporous silicon nitride background. The distribution of vesicle sizes(20-100 nm) is also suggestive that these particles are exosomes.

In some embodiments of the present invention, various coatings andlayers are applied to the nanoporous silicon nitride membrane. Forexample, very thin molecular layers with excellent hydrolytic stabilitymay be employed. For example, a layer of 1-10 nanometer thickness. Suchlayers are designed so as not to occlude the pores or reducepermeability of the membrane. Such coatings provide enhanced surfaceinteractions to assist in the capture of plasma components to supplementor otherwise interact with fluidic forces in the tangential flow deviceof the present invention.

An example of such a layer is that which is produced by functionalcarbene precursors to form uniform. Si—C and C—C attached monolayers onsilicon, silicon nitride, and inert organic polymers under mild vacuumconditions. By utilizing meta-stable carbene species generated undermild UV-light illumination, the activation barrier for the Si—C and C—Cbond formation is reduced and the variety of functional groups andsurfaces that can be modified through surface-grafting reactions isexpanded.

Ultrathin nanoporous silicon nitride (NPN) membranes can befunctionalized with stable and functional organic molecules via carbeneinsertion chemistry. One example of a suitable organic coating for NPNis a thin, inert polymer layer that serves as the carbene attachmentlayer, and a stable polyethylene glycol (PEG) terminated monolayer thatis linked to the polymer via non-hydrolytic C—C bonds generated by thevapor-phase carbene insertion. Such modifications to NPN provide thedesired organic functionalities without significantly impacting poresize distribution or transport properties.

Coatings and monolayers for a substrate such as nanoporous siliconnitride (NPN) that may be employed with the present invention aredescribed in U.S. patent application Ser. No. 15/130,208 to A.Shestopalov, L Xunzhi and J. L. McGrath filed on Apr. 15, 2016 andentitled “Methods for Depositing a Monolayer on a Substrate Field”, theentire disclosure of which is incorporated herein by reference in it'sentirety.

By defining surface chemistries, species capture from plasma can becontrolled and selective capture of plasma components can be realized.Different chemical handles can be used to functionalize NPN membranes.Mixtures of different chemical handles can be used to further modulatethe levels of adsorption of the plasma components and also to enhanceadsorption selectivity. These chemical handles can be used incombination with different tangential flow regimes and membrane poresizes to enhance specificity and selectivity of the membrane-plasmacomponent interactions.

In the device of the present invention, there are three distinctiveinterfaces between the nanoporous silicon nitride (NPN) and blood plasmathat act as non-binding, adsorbing, or selective surfaces for theselective removal of components such as extracellular vesicles.Individually these defined surfaces will (1) non-specifically limitadsorption of biomolecules from the plasma solution by creatingwater-like solvating environments near the interfaces (e.g.,polyethylene glycol molecules or zwitterionic species), (2)non-selectively enhance adsorption of various biomolecules through ionicinteractions and H-bonding (e.g., aminated interfaces), and (3)selectively bind serum components via specific biomolecular interaction(e.g., antigen-antibody interactions or specific H-bonding). Therefore,by creating homogeneously mixed monolayers that contain different ratiosof non-binding, adsorbing, and selective species, capture selectivitycan be established by the defined flow parameters and can further beenhanced by controlling the chemical composition of the membrane walls.

Defined surface chemistries may include, for example, antibodies thatcapture extracellular vesicles. Capture of extracellular vesicles byaffinity using antibodies may include tangential flow arrangements suchas those described and envisioned herein. In addition, antibodies may becombined with other defined surface chemistries for specificapplications. There are also antibodies that are specific toextracellular vesicles. For example, CD63, CD9, CD81 and Hsp70 all haveaffinity to exosomes. The present invention and the various embodimentsdescribed, depicted and envisioned herein includes generically theemployment of antibodies in general to capture, move, sort, retain, andotherwise process extracellular vesicles.

It is further stated that the various techniques, devices, methods andapparati described herein are also suitable for the capture of cell freeDNA. Cell free fetal DNA for example provides a rich population ofbiomarkers using maternal blood sampling as a form of non-invasiveprenatal diagnosis without the risk of procedure-related miscarriage.

The carbenylation approach can be used as a simple, robust and universalmethod to functionalize nanoporous materials with diverse classes oforganic and biological species. The inventors have demonstrated thatcarbenylated monolayers on Si, Ge, SiN, ITO and polymers can be modifiedwith various organic and biological molecules—small molecules,PEG-oligomers, GFP proteins and others—via simple surface reactions, andthat they exhibit excellent hydrolytic stability in water and aqueousbuffers for up to 2 weeks of exposure.

To form functional monolayers on nanoporous silicon nitride (NPN), themembranes will first be modified with an inert aliphatic coating thatserves as a passivating layer and as a carbene attachment interface.Subsequently, the NHS-diazirine carbene precursors will be used todeposit the NHS-terminated monolayers on the aliphatic coating throughthe thermodynamically and hydrolytically stable C—C bonds. Lastly,individual or mixed NHS-terminated molecules (non-binding, adsorbing,and selective) will be reacted with the NHS-terminated monolayer tomodify the resulting membranes with the desired chemicalfunctionalities.

FIG. 9 is an image and associated diagram depicting a nanoporous siliconnitride membrane supported on the edges and with a free standing,permeable region that has captured exosomes while the supported,impermeable region has captured no exosomes. This image is anotherexample of diffusion-driven physical sieving of extracellular vesiclesthat has been previously described and depicted herein.

FIG. 10 depicts EDX chemical analysis of an in use nanoporous siliconnitride membrane of the present invention. The region in the image showswhere salt crystals and exosomes are both present. The chemicalsignature of the spherical particles depicted do not contain NaCl peaks,confirming that they are not salt crystals.

Membranes with 100-1,000 nanometer diameter pores are fabricated withpatterning and etching methods. Specifically, 30 nanometer diameter poremembranes are fabricated using methods disclosed in PCT/US2014/1051316,the entire disclosure of which is incorporated herein by reference.

It can thus be seen that nanoporous silicon nitride membranes arecompatible with processing of high protein content biofluids, such asundiluted plasma, without evidence of membrane fouling. It can also beseen that the capture of extracellular vesicles within the pores ofnanoporous silicon nitride or similar membranes is a fast and elegantmethod for obtaining intact extracellular vesicles (such as exosomes) ofvery high purity. Further, the excess of nanoporous silicon nitridepores compared to the number of extracellular vesicles such as exosomessuggests that the present invention is able to capture nearly allexosomes in a biofluid sample.

A 30 nanometer pore size of nanoporous silicon nitride (NPN) membranesallows for the capture and retention of 30-100 nanometer extracellularvesicles such as exosomes, while passing contaminating species such as<30 nm proteins. In some embodiments of the present invention, largerpore sizes are desirable, and may include, for example, pore sizes aslarge as 120 nm. The large number of pores within our membranes(˜1.7×10⁸ pores/mm² assuming 35 nm pores and 16% porosity) exceeds thenumber of exosomes in most biofluids by several orders of magnitude(assuming 10⁵ exosomes/mL for plasma). This exosome-to-NPN pore ratiosuggests that nanoporous silicon nitride (NPN) membranes can capturenearly 100% of extracellular vesicles such as exosomes while leaving alarge number of pores unoccupied to enable the removal of smallercontaminants.

FIG. 11 is a diagram of fluid dynamics associated with exosome capture.The diagram depicts flow over a nanoporous silicon nitride membrane withexosomes represented as Brownian particles with diffusion coefficient Dand radius r. A given sample channel (height H; length L) is predictedto contain parabolic flow through the bulk with a slight permeation offluid through the membrane. Drag forces on the nanoparticles containedin the bulk will be largely tangential to the membrane, but very nearthe membrane they will be normal to the membrane as seen in FIG. 11.This region represents a capture layer with diffusion from the bulk intothis capture layer being the key physical process that must becontrolled by flow parameters.

Analytical techniques such as the creation of computational models forexosome capture can be used to determine the relationship between flowparameters and the capture of exosomes of various sizes. Computationalmodels may be built with finite element analysis software that includesmodeling of Brownian particles to the flow field. The models may, forexample, include the hydraulic permeability of ultrathin membranes andassume a Newtonian fluid with the viscosity of plasma. In any resultingmodel, fluid streamlines in the top sample channel are expected to beparabolic with a slight permeation through the membrane into the lowerchamber. The particles far from the channel will experience a large dragforce tangential to the membrane while those very close to the membranewill experience drag toward the membrane from transmembrane convectionand diminished tangential drag force. Exosomes entering this ‘capturelayer’ will be pulled into the pore of the membrane and held there solong as there is transmembrane pressure.

A computational model may predict, for example, the height of thecapture layer as a function of the flow parameters. It is expected thatmost well built computational models will indicate that the capturelayer will be very small compared to the channel height.

δ<<H

Thus it is only through diffusive excursions from the bulk to themembrane that most exosomes will become trapped in the membrane pores,and we can expect a Peclet defined as

${Pe} = {\frac{\overset{¯}{U}/L}{D/H^{2}} = \frac{\overset{¯}{U}H^{2}}{LD}}$

To be a key predictor of exosome capture. Note that because thediffusion coefficient and the drag forces imparted by the fluid on aparticle are both dependent on the friction factor f

f=6πρr

both will be dependent on the particle size r, and the probability ofcapture is expected to be strongly dependent on particle size. Use ofsuch modeling will allow one to prescribe flow settings that tune thecapture process to exosomes (or micro vessels) of a particular size. Useof such a model will allow determination of application specificdimensions to ensure complete capture of target particles (such asexosomes) from a flowable material in a single pass across the membraneof the present invention. Input pressures and channel dimensions are twosuch parameters. A computational model can also be used to prescribepressures during the recovery process if simple ‘backwashing’ provesproblematic in a given application and configuration. As previouslydescribed herein, defined surface chemistries may also be employed withthe membrane of the present invention for specific applications or toimprove the retention of desired material by the membrane, rejectnon-desired material, or remove the retained desired material whencertain conditions (such as a pressure change) are applied.

What is claimed is:
 1. A device for isolating extracellular vesiclesfrom biofluids, the device comprising: a nanoporous membrane comprisinga first surface with a plurality of pores; a tangential fluid flowdevice for creating a tangential fluid flow velocity of a biofluidacross the surface of the nanoporous membrane having a plurality ofpores; and a pressure gradient device for creating a pressure gradientthrough the nanoporous membrane.
 2. The device of claim 1, furthercomprising a device for reversing the pressure gradient through thenanoporous membrane to release captured extracellular vesicles.
 3. Thedevice of claim 1, wherein the nanoporous membrane is nanoporous siliconnitride.
 4. The device of claim 1, wherein the density of pores of thenanoporous membrane is at least 10⁷ pores per square millimeter.
 5. Thedevice of claim 1, wherein the range of pore diameters in the nanoporousmembrane is on the average between 20 nanometers and 120 nanometers. 6.The device of claim 1, wherein the magnitude of the created tangentialflow velocity is between 100 micrometers per second and 10 centimetersper second.
 7. The device of claim 1, wherein the magnitude of thecreated pressure gradient through the nanoporous membrane is between 1pascal and 1 atmosphere.
 8. The device of claim 1, wherein thenanoporous membrane is configured as a channel having a channel lengthand a channel height for containing tangential flow of a biofluid. 9.The device of claim 8, wherein the channel length along the principaldirection of flow is between 1 millimeter and 1 meter.
 10. The device ofclaim 8, wherein the channel height is between 100 nanometers and 1millimeter.
 11. The device of claim 1, wherein the nanoporous membranefurther comprises a coating.
 12. The device of claim 1, wherein thenanoporous membrane further comprises a defined surface chemistry. 13.The device of claim 1, wherein the nanoporous membrane further comprisesa carbenylated monolayer.
 14. The device of claim 1, wherein thenanoporous membrane further comprises an aliphatic coating.
 15. Thedevice of claim 1, wherein the nanoporous membrane further comprisespolyethylene glycol.
 16. The device of claim 1, wherein the nanoporousmembrane further comprises a zwitterionic species.
 17. The device ofclaim 1, wherein the nanoporous membrane further comprises an aminatedinterface.
 18. The device of claim 1, wherein the nanoporous membranefurther comprises a second surface where the plurality of pores from thefirst surface extend through the second surface and where the pluralityof pores of the first surface are of a greater diameter than the relatedplurality of pores that extend through the second surface.
 19. A methodfor isolating extracellular vesicles from biofluids, the methodcomprising the steps of: providing a biofluid; passing the biofluid overa nanoporous membrane in a tangential flow orientation wherein thenanoporous membrane comprises a plurality of pores; and creating apressure gradient through the nanoporous membrane during tangential flowof the biofluid to capture extracellular vesicles contained within thebiofluid.
 20. The method for isolating extracellular vesicles frombiofluids as stated in claim 19, the method further comprising the stepof: reversing the pressure gradient through the nanoporous membrane toelute the captured extracellular vesicles from the nanoporous membraneonce a specified volume of biofluid has passed.
 21. The method forisolating extracellular vesicles from biofluids as stated in claim 19,wherein the nanoporous membrane is nanoporous silicon nitride.
 22. Themethod for isolating extracellular vesicles from biofluids as stated inclaim 19, wherein the nanoporous membrane further comprises a definedsurface chemistry.
 23. A device for isolating cell free DNA frombiofluids, the device comprising: a nanoporous membrane comprising asurface with a plurality of pores; is a tangential fluid flow device forcreating a tangential fluid flow velocity of a biofluid across thesurface of the nanoporous membrane having a plurality of pores; and apressure gradient device for creating a pressure gradient through thenanoporous membrane.
 24. The device of claim 23, further comprising adevice for reversing the pressure gradient through the nanoporousmembrane to release captured cell free DNA.
 25. The device of claim 23,wherein the nanoporous membrane is nanoporous silicon nitride.
 26. Thedevice of claim 23, wherein the density of pores of the nanoporousmembrane is at least 10⁷ pores per square millimeter.
 27. The device ofclaim 23, wherein the range of pore diameters in the nanoporous membraneis on the average between 20 nanometers and 120 nanometers.
 28. Thedevice of claim 23, wherein the magnitude of the created tangential flowvelocity is between 100 micrometers per second and 10 centimeters persecond.
 29. The device of claim 23, wherein the magnitude of the createdpressure gradient through the nanoporous membrane is between 1 pascaland 1 atmosphere.
 30. The device of claim 23, wherein the nanoporousmembrane is configured as a channel having a channel length and achannel height for containing tangential flow of a biofluid.
 31. Thedevice of claim 30, wherein the channel length along the principaldirection of flow is between 1 millimeter and 1 meter.
 32. The device ofclaim 30, wherein the channel height is between 100 nanometers and 1millimeter.
 33. The device of claim 23, wherein the nanoporous membranefurther comprises a coating.
 34. The device of claim 23, wherein thenanoporous membrane further comprises a defined surface chemistry. 35.The device of claim 23, wherein the nanoporous membrane furthercomprises a carbenylated monolayer.
 36. The device of claim 23, whereinthe nanoporous membrane further comprises an aliphatic coating.
 37. Thedevice of claim 23, wherein the nanoporous membrane further comprisespolyethylene glycol.
 38. The device of claim 23, wherein the nanoporousmembrane further comprises a zwitterionic species.
 39. The device ofclaim 23, wherein the nanoporous membrane further comprises an aminatedinterface.
 40. A method for isolating cell free DNA from biofluids, themethod comprising the steps of: providing a biofluid; passing thebiofluid over a nanoporous membrane in a tangential flow orientationwherein the nanoporous membrane comprises a plurality of pores; andcreating a pressure gradient through the nanoporous membrane duringtangential flow of the biofluid to capture cell free DNA containedwithin the biofluid.
 41. The method for isolating cell free DNA frombiofluids as stated in claim 40, the method further comprising the stepof: reversing the pressure gradient through the nanoporous membrane toelute the cell free DNA from the nanoporous membrane once a specifiedvolume of biofluid has passed.
 42. The method for isolating cell freeDNA from biofluids as stated in claim 40, wherein the nanoporousmembrane is nanoporous silicon nitride.
 43. The method for isolatingcell free DNA from biofluids as stated in claim 40, wherein thenanoporous membrane further comprises a defined surface chemistry.